Publications

The global pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to efforts in developing effective vaccine approaches. Currently, approved coronavirus disease 2019 (COVID-19) vaccines are administered through an intramuscular (I.M.) route. Here, we show that the SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD), when displayed on immunogenic liposomes, can be intranasally (I.N.) administered, resulting in the production of antigen-specific IgA and antigen-specific cellular responses in the lungs. Following I.N. immunization, antigen-presenting cells of the lungs took up liposomes displaying the RBD. K18 human ACE2-transgenic mice that were immunized I.M or I.N with sub-microgram doses of RBD liposomes and that were then challenged with SARS-CoV-2 had a reduced viral load in the early course of infection, with I.M. achieving complete viral clearance. Nevertheless, both vaccine administration routes led to full protection against lethal viral infection, demonstrating the potential for the further exploration and optimization of I.N immunization with liposome-displayed antigen vaccines.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to NF-κB activation and induction of pro-inflammatory cytokines, though the underlying mechanism for this activation is not fully understood. Our results reveal that the SARS-CoV-2 Nsp14 protein contributes to the viral activation of NF-κB signaling. Nsp14 caused the nuclear translocation of NF-κB p65. Nsp14 induced the upregulation of IL-6 and IL-8, which also occurred in SARS-CoV-2 infected cells. IL-8 upregulation was further confirmed in lung tissue samples from COVID-19 patients. A previous proteomic screen identified the putative interaction of Nsp14 with host Inosine-5'-monophosphate dehydrogenase 2 (IMPDH2), which is known to regulate NF-κB signaling. We confirmed the Nsp14-IMPDH2 protein interaction and identified that IMPDH2 knockdown or chemical inhibition using ribavirin (RIB) and mycophenolic acid (MPA) abolishes Nsp14- mediated NF-κB activation and cytokine induction. Furthermore, IMPDH2 inhibitors (RIB, MPA) or NF-κB inhibitors (bortezomib, BAY 11-7082) restricted SARS-CoV-2 infection, indicating that IMPDH2-mediated activation of NF-κB signaling is beneficial to viral replication. Overall, our results identify a novel role of SARS-CoV-2 Nsp14 in inducing NF-κB activation through IMPDH2 to promote viral infection.

Intranasal vaccination offers the potential advantage of needle-free prevention of respiratory pathogens such as influenza viruses with induction of mucosal immune responses. Optimal design of adjuvants and antigen delivery vehicles for intranasal delivery has not yet been well established. Here, we report that an adjuvant-containing nanoliposome antigen display system that converts soluble influenza hemagglutinin antigens into nanoparticles is effective for intranasal immunization. Intranasal delivery of nanoliposomes in mice delivers the particles to resident immune cells in the respiratory tract, inducing a mucosal response in the respiratory system as evidenced by nasal and lung localized IgA antibody production, while also producing systemic IgG antibodies. Intranasal vaccination with nanoliposome particles decorated with nanogram doses of hemagglutinin protected mice from homologous and heterologous H3N2 and H1N1 influenza virus challenge. IMPORTANCE A self-assembling influenza virus vaccine platform that seamlessly converts soluble antigens into nanoparticles is demonstrated with various H1N1 and H3N2 influenza antigens to protect mice against influenza virus challenge following intranasal vaccination. Mucosal immune responses following liposome delivery to lung antigen-presenting cells are demonstrated.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause lethal pulmonary damage in humans. It contains spike proteins on its envelope that bind to human angiotensin-converting enzyme 2 (hACE2) expressed on airway cells, enabling entry of the virus, and causing infection. The soluble form of hACE2 binds SARS-CoV-2 spike protein, prevents viral entry into target cells, and ameliorates lung injury; however, its short half-life limits therapeutic utilities. Here, synthetic mRNA is engineered to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. A novel lipid nanoparticle (LNP) is used for packaging and delivering mRNA to cells to produce hsACE2 proteins. Intravenously administered LNP delivers mRNA to hepatocytes, leading to the production of circulatory hsACE2 initiated within 2 h and sustained over several days. Inhaled LNP results in lung transfection and secretion of mucosal hsACE2 to lung epithelia, the primary site of entry and pathogenesis for SARS-CoV-2. Furthermore, mRNA-generated hsACE2 binds to the receptor-binding domain of the viral spike protein. Finally, hsACE2 effectively inhibits SARS-CoV-2 and its pseudoviruses from infecting host cells. The proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed to neutralize SARS-CoV-2 and open new treatment opportunities for coronavirus disease 2019 (COVID-19).

PA-X is a nonstructural protein of influenza A virus (IAV), which is encoded by the polymerase acidic (PA) N-terminal region that contains a C-terminal +1 frameshifted sequence. IAV PA-X protein modulates virus-induced host innate immune responses and viral pathogenicity via suppression of host gene expression or cellular shutoff, through cellular mRNA cleavage. Highly pathogenic avian influenza viruses (HPAIV) of the H5N1 subtype naturally infect different avian species, they have an enormous economic impact in the poultry farming, and they also have zoonotic and pandemic potential, representing a risk to human public health. In the present study, we describe a novel bacterium-based approach to identify amino acid residues in the PA-X protein of the HPAIV A/Viet Nam/1203/2004 H5N1 that are important for its ability to inhibit host protein expression or cellular shutoff activity. Identified PA-X mutants displayed a reduced shutoff activity compared to that of the wild-type A/Viet Nam/1203/2004 H5N1 PA-X protein. Notably, this new bacterium-based screening allowed us to identify amino acid residues widely distributed over the entire N-terminal region of PA-X. Furthermore, we found that some of the residues affecting A/Viet Nam/1203/2004 H5N1 PA-X host shutoff activity also affect PA polymerase activity in a minigenome assay. This information could be used for the rational design of new and more effective compounds with antiviral activity against IAV. Moreover, our results demonstrate the feasibility of using this bacterium-based approach to identify amino acid residues important for the activity of viral proteins to inhibit host gene expression. IMPORTANCE Highly pathogenic avian influenza viruses continue to pose a huge threat to global animal and human health. Despite of the limited genome size of Influenza A virus (IAV), the virus encodes eight main viral structural proteins and multiple accessory nonstructural proteins, depending on the IAV type, subtype, or strain. One of the IAV accessory proteins, PA-X, is encoded by the polymerase acidic (PA) protein and is involved in pathogenicity through the modulation of IAV-induced host inflammatory and innate immune responses. However, the molecular mechanism(s) of IAV PA-X regulation of the host immune response is not well understood. Here, we used, for the first time, a bacterium-based approach for the identification of amino acids important for the ability of IAV PA-X to induce host shutoff activity and describe novel residues relevant for its ability to inhibit host gene expression, and their contribution in PA polymerase activity.

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 103 and 2 × 104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 101 and 2 × 102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.

The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4′-fluorouridine (4′-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4′-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.

Influenza A viruses (IAVs) infect a broad range of hosts, including multiple avian and mammalian species. The frequent emergence of novel IAV strains in different hosts, including in humans, results in the need for vigilance and ongoing development of new approaches to fighting or prevent those infections. Canine influenza is a contagious respiratory disease in dogs caused by two subtypes of IAV, the equine-origin H3N8 canine influenza virus (CIV), and the avian-origin H3N2 CIV. A novel approach to influenza vaccination involves single-cycle infectious influenza A viruses (sciIAVs), which are defective for an essential viral gene. They are propagated in complementing cell lines which provide the missing gene in trans. As sciIAV cannot complete their replication cycle in regular cells they are limited to a single round of viral replication. Because of their safety profile and ability to express foreign antigens inside infected cells, sciIAVs have served both as live-attenuated vaccines and as vaccine vectors for the expression of heterologous antigens. Here, we describe experimental procedures for the generation of a single-cycle infectious CIV (sciCIV), where the viral hemagglutinin (HA) gene was exchanged for the gene for green fluorescent protein (GFP). Complementation of the viral HA protein is provided in trans by stable HA-expressing cell lines. Methods for the in vitro characterization of HA deficient but GFP-expressing sciCIV (sciCIV ΔHA/GFP) are described, as well as its use as a potential vaccine.

Reporter-expressing recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) represents an excellent tool to understand the biology of and ease studying viral infections in vitro and in vivo. The broad range of applications of reporter-expressing recombinant viruses is due to the facilitated expression of fluorescence or bioluminescence readouts. In this chapter, we describe a detailed protocol on the generation of rSARS-CoV-2 expressing Venus, mCherry, and NLuc that represents a valid surrogate to track viral infections.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.